Abstract Autoimmune diseases occur when immune cells escape protective mechanisms that prevent their activation in response to the organism's own tissues. Co-inhibitory receptors such as PD-1, Tim-3 and TIGIT, expressed on activated T cells, are a crucial mechanism to control inappropriate T cell activation and protect tissues from autoimmune attack. Systemic Sclerosis (SSc, Scleroderma) is a chronic autoimmune disease where dysregulated immune cells contribute to the initiation and/or progression of microvascular damage and fibrosis, leading to devastating tissue malfunction, with high morbidity and mortality. While autoreactive T cells have been detected in SSc, their role in disease pathogenesis is poorly understood. To gain insight in the phenotype and function of T cells from SSc patients, we compared the expression of multiple co-inhibitory receptors on PBMCs from SSc patients vs healthy controls. Interestingly, various subsets of T cells from SSc patients showed increased expression of these receptors, consistent with the presence of a state of chronic immune activation and attempts by the organism to control autoreactivity and inflammation. Surprisingly however, blocking co-inhibitory receptors during in vitro stimulation of SSc PBMCs did not uniformly increase cytokines but altered complex expression patterns, as revealed by multiplex ELISA and computational modeling. These findings provide the premise for this grant, in which we propose to determine how co-inhibitory receptors differentially regulate cytokine profiles in SSc vs healthy T cells and how these aberrantly controlled cytokines promote fibrosis. In Aim 1, we will characterize T cell populations that show increased expression of co-inhibitory receptors. We will test the functional significance of this altered expression by stimulating PBMCs, as well as isolated T cell subsets, in vitro with dendritic cells carrying the ligands for the co-inhibitory receptors. Addition of co-inhibitory receptor-blocking antibodies to the cultures will reveal their role in the production of pro- and anti-fibrotic cytokines. Finally, we will corroborate the relevance of our findings for skin T cells from SSc patients. In Aim 2, we will measure the impact of enhanced co-inhibitory receptor expression on the interaction of T cells with two cell types that are critical in SSc pathology: fibroblasts and endothelial cells. Fibroblast and endothelial differentiation and function will be followed in the target cells after co-culture with SSc T cells in the absence and presence of reagents modulating co-inhibitory function. Understanding how T cell dysfunction impacts the crosstalk between different cellular players in the complex fibrotic process may reveal novel pathways accessible for therapeutic intervention in SSc.